Long-chain polyunsaturated fatty acids (PUFAs) have been implicated in human brain development as well as in the maintenance of cardiovascular health. Although animals have the enzymes necessary to form long-chain PUFAs through the elongation of plant-derived PUFAs, this oxygen-dependent process is not efficient. An efficient pathway for the biosynthesis of PUFAs in deep-sea bacteria utilizes a polyketide synthase-like (PKS-like) multienzyme complex. A total of five genes from this pathway have been found to be sufficient for the production of polyunsaturated fatty acids in an otherwise non-producing Escherichia coli. These genes are pfaA, pfaB, pfaC, pfaD, encoding PUFA synthases containing enzyme domains for acyl tranferases (AT), keto-acyl synthase (KS), acyl carrier protein (ACP), keto-acyl reductase (KR), enoyl reductase (ER) and dehydratase (DH) activities and also pfaE, which encodes a required phosphopantetheine transferase (PPTase) essential for the activation of ACP domains through chemical modification as shown in FIG. 1. While some of the required enzymatic activities are housed in independent stand-alone proteins (pfaB, pfaD and pfaE: FIG. 1) others are assembled into multidomains (pfaA and pfaC: FIG. 1). No thioesterase activity has been observed in the PUFA synthase cluster and no dedicated thioesterase protein from the producing organism is required for heterologous production of PUFAs in E. coli. 
Dehydratase (DH) domains are responsible for the formation of the cis double bonds in the structure of PUFAs. They can be easily identified by their sequence similarity to FabA and FabZ, the two DH enzymes involved in fatty acid biosynthesis in E. coli. FabA/Z catalyze the dehydration of 3Rhydroxyacyl-ACP via a syn elimination mechanism which has also been reported in the DH domain from the erythromycin PKS.
The structure of FabA, and more recently FabZ, revealed an obligate homodimeric arrangement in which both DH subunits contribute key residues to the active site. This distinct architectural feature has been found to extend to DH domains from the animal Fatty Acid Synthase (FAS), and more recently to the erythromycin PKS, although with the following variation on the E. coli arrangement. While the E. coli FabA and FabZ form homodimers of identical subunits, the DH domains from FAS and PKS systems form a heterodimeric double hotdog arrangement in which two contiguous pseudosubunits are housed within the same polypeptide and separated by a 25-residue amino acid stretch. Thus, the required dimerization of the DH domain in the context of a multienzyme complex does not necessarily involve interactions between different polypeptides, but rather within the same polypeptide.
In both the FAS and PKS DH, the protein region that is homologous to FabA is followed by a necessary C-terminal pseudodomain with no previously known function and no known sequence homologue. In the case of the FAS DH, the C-terminal pseudodomain was found to contribute to dehydratase activity in in vitro enzyme assays. The structure of the PKS DH showed that the Cterminal pseudodomain forms the other half of the double hotdog in the three-dimensional structure. In that work, the protein construct that was crystallized, and whose structure was determined, contained the pseudodomain but lacked dehydratase activity in vitro, although mutations made elsewhere did show an effect on overall polyketide production by the full-length multienzyme.
The PUFA synthase multienzyme contains two putative DH domains in tandem. They have been identified as DH domains based on their sequence similarity to FabA/Z, but their activity or specificity has not been confirmed biochemically. The tandem arrangement, while not previously observed in other biosynthetic enzyme systems, is a well-conserved feature of PUFA synthases. However, it is unknown how these tandem domains act to generate the combination of double and single C—C bonds in the final PUFA structure.